Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by a deficiency of enzymes involved in cortisol biosynthesis. More than 90% of the cases are due to 21-hydroxilase deficiency. This leads to the accumulation of 17-hydroxyprogesterone (17OHP) and allows diagnosis of this disorder in newborn screening (NS) programs. Premature newborns typically have higher levels than full-term newborns, making the use of age-appropriate cut-off values important for accurate interpretation. The aim of our study was to update the gestational age-adjusted cut-off values for 17OHP, the methodology currently used in Argentina by the jurisdictions participating in the National Program for the Strengthening of Early Detection of Congenital Diseases. Cut-off limits were obtained using percentiles of the distribution of 17OHP values for each gestational age on 53,230 dry blood spot (DBS) samples. Sensitivity, specificity, false positive rate, positive predictive value, and incidence were calculated. We successfully established gestational age-specific cut-off values for 17OHP. The analysis showed a sensitivity of 100%, specificity of 99.52%, a false positive rate of 0.49%, and a positive predictive value of 1.54%. The incidence of CAH was 1:13,308 live births. These findings underscore the importance of using population-appropriate cut-off limits. Such harmonization enables comparability among regional neonatal screening programs with the added benefit of decreasing the rate of recalls and associated costs.